Project Summary/Abstract Beta-2 glycoprotein-I (b2GPI) is a 50-kDa glycoprotein that circulates in the blood at a concentration of 0.2 mg/ml. It was originally discovered in 1961 but received little attention until 1990 when it was identified as the dominant antigen of antiphospholipid antibodies (aPL) in the autoimmune disorder known as Antiphospholipid Syndrome (APS). Patients affected by APS develop blood clots in veins and arteries as well as pregnancy complications. Occlusions of the vascular system may lead to life-threatening complications such as myocardial infarction, pulmonary embolism and stroke. Despite the established correlation between the presence of anti-b2GPI antibodies and thrombosis, APS remains incredibly challenging to diagnose and treat for physicians. There are two main issues. First, anti-b2GPI antibodies are very heterogeneous, and not all of them are pathogenic. Hence, laboratory tests are difficult to develop, standardize and interpret. This affects our ability to identify, with confidence, patients at higher risk for thrombosis who require prompt pharmacological intervention. Second, treatment options are limited to the prophylactic administration of long-term anticoagulants, anti-platelets, and low molecular weight heparin, that block the downstream effect of aPL, i.e., activation of the clotting cascade. This mechanism of action, however, is non-optimal for treating APS patients and, in addition to negatively affecting patients? lifestyle and exposing them to the risk of fatal bleeding, among other things, these drugs also fail to prevent thrombotic recurrences in ~30% of the APS patient population, especially in those individuals with arterial thrombosis. To address these unmet clinical needs, physician and basic scientists have joined forces over the past twenty years and launched several key initiatives with the goal of standardizing the diagnosis of APS patients and determining the optimal management of aPL-positive patients. Thus, understanding the role of b2GPI in APS became a top priority in the field. Thanks to these collective efforts, much has been learned about the subclasses of aPL causing thrombosis and the signaling pathways triggered by anti-?2GPI antibodies. Less clear, however, remain the structural properties of ?2GPI, the mechanisms controlling antigen-antibody recognition and the circumstances under which ?2GPI becomes immunogenic. To bridge this gap in our fundamental knowledge, this research project seeks to elucidate the structural architecture ?2GPI under conditions relevant to physiology, define the structural determinants for the interaction of ?2GPI with aPL and physiological ligands, and develop an improved diagnostic test for the detection of pathogenic anti-b2GPI antibodies in patients? plasma. Information gathered through these studies will contribute to establishing the missing link between structure, function, and immunogenicity of ?2GPI in APS, and will open new avenues for APS-specific diagnostics and therapeutics.